Metabolomics approach to assess the effect of siphonal autotomy on metabolic characteristics of razor clam Solen grandis

Autotomy appendages are fundamental evolutionary adaptations to escape predation. The siphon is an important foraging organ for bivalves. Here, we report the first demonstration of autotomy of the siphon in marine bivalves (razor clam Solen grandis) and the effect of siphonal autotomy in S. grandis on foraging and metabolic characteristics. In this study, the feeding rate and digestive enzyme activities upon siphonal autotomy in razor clams were investigated. Moreover, endogenous metabolites pre/post-autotomy of the siphon were investigated using liquid chromatography tandem-mass spectrometry (LC–MS). The feeding rate and digestive enzyme activities decreased significantly after siphonal autotomy in S. grandis (P < 0.05), suggesting that autotomy of the siphon negatively affected its foraging. These results might be related to the reduction in the foraging radius. Additionally, the effect of autotomy was investigated on a total of 34 differentially abundant metabolites, and pathway analysis indicated that 32 differentially enriched metabolic pathways were worthy of attention. Further integrated key metabolic pathway analysis showed that glycine, serine and threonine metabolism; taurine and hypotaurine metabolism; biotin metabolism; vitamin B6 and thiamine metabolism were significantly relevant pathways in S. grandis pre/post-autotomy of the siphon. The downregulation of glycine, taurine, and hypotaurine is expected to indicate a shortage of intermediate compounds and energy in S. grandis. Therefore, to provide the required energy and materials for siphon regeneration in S. grandis, we anticipated that it would be necessary to supplement these as exogenous metabolites from the daily diet.

www.nature.com/scientificreports/ addition, collision and shaking may cause razor clams to autotomize siphons under stimulated conditions during transportation and sales, causing the razor clams to lose body weight and price. However, the effects of siphonal autotomy and the underlying metabolic characteristics of S. grandis remain poorly understood. Metabolomics is an emerging technological and analytical approach for comprehensively analyzing the various metabolites contained in biological samples 21,22 . Metabolites comprise all compounds in a biological matrix that are typically smaller than 1 kDa in size and include small peptides, oligonucleotides, sugars, organic acids, ketones, aldehydes, amino acids, lipids, steroids, alkaloids and xenobiotics 23,24 . With the development of metabolomics technology, many tools are currently available, including liquid chromatography tandem-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) 23 . However, compared with GC-MS, LC-MS has a high peak capacity, resolution, and sensitivity. It is suitable for the analysis of metabolites with a high boiling point, high molecular weight or limited thermal stability 23,25 . Thus, LC-MS is highly suitable for the detection of a wide array of metabolites. To date, LC-MS metabolomics have been applied to metabolic studies of bivalves. For instance, Tian et al. reported that the metabolic characteristics of live scallops (Mizuhopecten yessoensis) subjected to mechanical shock were investigated in the early post-harvest process 22 . Sun et al. revealed the molecular responses of clams to acute hypoxia by combining integrated transcriptome and metabolome (LC-MS) analysis 26 . Abraham et al. identified biomarkers of brevetoxin exposure in hard clams (Mercenaria sp.) exposed to Karenia brevis blooms using LC-MS 27 . However, to the best of our knowledge, there are no studies that have investigated the metabolic characteristics of S. grandis.
In this study, we investigated the feeding rate and digestive enzyme activities upon siphonal autotomy in razor clams. Moreover, using LC-MS, we explored the changes in endogenous metabolites pre/post-autotomy of the siphon. The results contribute to data on the underlying metabolic characteristics and toward improving aquaculture of S. grandis.

Materials and methodology
Sample collection and preparation. Razor clams were obtained from the Jiangsu Fine Breeding Farm of Solen grandis (Nantong, China). A total of 18 individuals were randomly selected, including 9 razor clams with induced autotomy of the siphon and 9 razor clams with a complete siphon. Three individuals were placed in one aquarium were considered a single sample. Therefore, a total of six samples were divided into two groups: three pre-autotomy groups and three post-autotomy groups. The feeding rates of S. grandis were assessed by measuring the volume of water cleared from suspended microalgae (Isochrysis galbana) per hour 28 . At the end of the feeding experiment, we measured body weight and dissected the digestive glands and siphon base separately for each sample. Each sample was washed with 0.01 M phosphate buffered saline, immediately frozen in liquid nitrogen, and stored at − 80 ℃ for subsequent metabolite isolation and bioactivity determination.
Determination of feeding rates and digestive enzyme activity assay. The feeding rate was measured by means of the clearance method using an aquarium with three S. grandis and well-mixed seawater with added algal cells (Isochrysis galbana) that are 100% efficiently retained by the gills of S. grandis 28 . The algal concentration in seawater was measured using a hemocytometer both at the beginning and end (1 h later) of the experiment. The feeding rate was determined from the exponential decrease in algal concentration as a function of time.
The digestive glands were weighed and homogenized in ice-cold 0.86% sterile saline solution (tissue: saline, 1:9). Then, the homogenates were centrifuged at 1200 g at 4 ℃ for 15 min, and the supernatants were collected and stored at − 80 ℃ for the analysis of digestive enzymes. The activities of digestive enzymes, including protease, amylase, and lipase, were examined spectrophotometrically using commercial assay kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer's instructions 29 . All assays were performed in triplicate. In the reaction, protease hydrolyzes proteins to produce phenolic amino acids, and phenolic reagents can be reduced to blue substances by phenolic amino acids. Thus, one unit of protease was defined as the production of 1 μg of amino acid per milligram of histone per minute at 37 °C. Amylase can hydrolyze starch to generate glucose, maltose, and dextrin. In the case of known and excessive substrate concentrations, iodine solution is added to combine with unhydrolyzed starch to generate a blue complex. Amylase activity was calculated according to the depth of blue color. One amylase activity unit was defined as the hydrolysis of 10 mg of starch per mg protein at 37 °C for 30 min. Latex made of triglycerides and water has opacification properties owing to the absorption and scattering of incident light by micelles. Under the action of lipase, the triglycerides in micelles hydrolyze, causing micelles to split, thus reducing the scattering light or turbidity. The rate of decrease is related to lipase activity. One unit of lipase was defined as each gram of tissue protein reacting with each substrate consumed 1 μmol substrate per minute in the reaction system at 37 °C. It is worth noting that the activities of digestive enzymes in this study are presented as specific activities.

Metabolite extraction and analysis for LC-MS.
We dissected the siphon base tissue to measure the metabolites of the razor clam S. grandis. Twenty-five milligrams of sample were weighed and placed into a clean microcentrifuge tube, and 500 µL of extraction solution (acetonitrile: methanol: water = 2: 2: 1) containing isotopically labeled internal standard mixture was added. After vortexing for 30 s, the samples were homogenized at 35 Hz for 4 min (JXFSTPRP-24; Shanghai Jingxin Technology Co., Ltd., China) and sonicated for 5 min in an ice-water bath (YM-080S, Shenzhen Fangao Microelectronics Co., Ltd., China). Homogenization and sonication cycles were repeated for twice. The samples were incubated at − 40 ℃ for 1 h and centrifuged at 13,800 g for 15 min at 4 ℃ (Heraeus Fresco17 series; Thermo Fisher Scientific Inc., USA). The supernatant (400 μL) was transferred to a fresh tube and dried in a vacuum concentrator at 37 ℃. Then, the dried samples were reconstituted in 200 μL of 50% acetonitrile by sonication for 10 min in ice-water bath. The mixture was then centrifuged Triple TOF 6600 mass spectrometry (AB SCIEX) was used for its ability to acquire MS/MS spectra on an information-dependent basis during an LC-MS experiment. In this mode, the acquisition software (Analyst TF 1.7; AB Sciex) continuously evaluates the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on preselected criteria. In each cycle, the most intensive 12 precursor ions with intensity above 100 were chosen for MS/MS at a collision energy of 30 eV. The cycle time was 0.56 s. The ESI source conditions were set as following: Gas 1 as 60 psi, Gas 2 as 60 psi, curtain gas as 35 psi, source temperature as 600 ℃, declustering potential as 60 V, ion spray voltage floating as 5000 V or − 4000 V in positive or negative modes, respectively. Statistical analysis. The MS raw data (.wiff) files were converted to the mzXML format using ProteoWizard, and processed using R package XCMS 30 . The process includes peak deconvolution, alignment and integration. Minfrac and cut off were set as 0.5 and 0.3 respectively. The in-house MS2 database was used for metabolite identification. The significance of the differences between groups was determined using Student' s t-test. Body weight, feeding rate, and digestive enzyme activities are presented as the mean ± SD.

Results
Comparison of feeding rate and digestibility. The body weight was reduced by 4.83 ± 0.85 g and the feeding rate of S. grandis was down-regulated significantly (P = 0.035) after siphonal autotomy (Fig. 1). To further understand the effect of siphonal autotomy on feeding capacity, digestive enzyme activity levels were measured. The study of amylase, lipase, and proteinase activities pre/post-autotomy of the siphon can help us understand the food digestion capacity of S. grandis. In this study, digestive enzyme activity levels in the post-autotomy group were significantly lower than in the pre-autotomy group (P < 0.05), suggesting that the digestibility in S. grandis was significantly inhibited after autotomy (Fig. 2).
Metabolic profiles analyzed by LC-MS. All metabolites were analyzed using unsupervised principal component analysis (PCA). PCA score results showed that the pre/post-autotomy data points were significantly separated in spatial distribution and the R 2 X values of the PCA model accounting for the variance were 0.469 and 0.958 in the positive and negative ion modes, respectively (Fig. 3a,b). To maximize the discrimination between the two groups, we employed orthogonal projections to latent structures-discriminant analysis (OPLS- www.nature.com/scientificreports/ DA) to elucidate the different metabolic patterns. The OPLS-DA results show that data points between the two groups were divided into separate clusters, suggesting that metabolic patterns pre/post-autotomy of the siphon in S. grandis were significantly different (Fig. 3c,d). All samples in the score plots were within the 95% Hotelling's T-squared ellipse in PCA and OPLS-DA, thereby indicating that there were no outliers among the analyzed samples and might be exploited in subsequent analyses. Volcano plots were used to illustrate the relationships between the P-value and fold change of all the identified metabolites, representing the degree of difference and the statistical significance in the pre/post autotomy groups in S. grandis. The volcano plots showed that 34 significantly differential metabolites (SDMs) (VIP > 1 and P < 0.05) were determined pre/post autotomy of the siphons in S. grandis, based on the OPLS-DA results. Among these SDMs, 22 differential metabolites were identified based on the positive ion mode, including seven upregulated metabolites and 15 downregulated metabolites (Fig. 5a). Based on the negative ion mode, 12 differential metabolites were identified, including five upregulated metabolites and seven downregulated metabolites (Fig. 5b). These SDMs include carbohydrate metabolites (e.g., ADP-ribose, and ADP-glucose), amino acids and their derivatives (e.g., glycine, asparagine, and alanine.), lipid metabolites (e.g., dethiobiotin, methyl acetoacetate, and hypotaurine), and other metabolites (e.g., tetramisole, and thiabendazole) (Fig. 6).
Characterization and functional analysis of key metabolic pathways of significantly differential metabolites. SDMs were imported into MetaboAnalyst 4.0 and KEGG pathway analysis was performed to identify the potential metabolic pathways that are affected upon autotomy of the siphon in S. grandis 31 . A total of 32 pathways (POS:26, NEG:6) were identified (Fig. 7). We constructed a schematic overview based on the reference diagrams stored in the KEGG database 32,33 (Fig. 8). Base on both the enrichment factor and pathway name, the relevant metabolic pathways were identified as vitamin B6 and thiamine metabolism; alanine, aspartate and glutamate metabolism; starch and sucrose metabolism; and taurine and hypotaurine metabolism (Fig. 7). We summarized these different enriched metabolic pathways as the metabolism of amino acids, carbohydrates, nucleosides, vitamins and cofactors. www.nature.com/scientificreports/

Discussion
Autotomy (self-amputation) may negatively affect competition, mobility, mating and foraging [3][4][5][6] . The siphon is an important foraging organ for bivalves. In this study, the feeding rate and digestion capacity decreased after siphonal autotomy in S. grandis, which may be related to the reduction in foraging radius. However, the material changes and accumulation in these bivalves remain poorly understood. Importantly, determination of the metabolite changes inside the cells after autotomy of the siphon will help us provide new insights into the underlying metabolic characteristics and help improve aquaculture of S. grandis. In total, 32 major pathways were observed pre/post-autotomy of the siphon in S. grandis, of which amino acid metabolism was significant and played a significant role in autotomy. Glycine is regarded as a key link to one-carbon metabolism and is involved in the methylation of proteins and deoxyribonucleic acid 34 . It is also a fundamental component of the biosynthesis of heme, purines, creatine, glutathione, and uric acid 35 . The relatively low content of glycine after autotomy of the siphon could be related to L-alanine, L-aspartate, and L-methionine www.nature.com/scientificreports/ metabolism. These nitrogenous metabolites are further converted into intermediates of the TCA cycle to compensate for the shortage of energy and intermediate compounds (e.g., Cys-Ala, and Ala-Thr). Taurine and hypotaurine were the two SDMs in the TCA cycle in this study. Taurine is known to maintain cell membrane permeability in calcium homeostasis 36 . Taurine can also remove oxidizing free radicals and plays a strong antioxidant role in detoxification and osmoregulation 37 . When the razor clam's siphon undergoes autotomy, a large amount of oxidized free radicals may be produced. However, the razor clam has the ability to respond to autotomy by changing taurine and hypotaurine metabolism. The TCA cycle has two important functions. The first is an intermediate compound that synthesizes amino acids and fatty acids. The second involves the formation of large amounts    39,40 . Therefore, it is necessary to supplement with exogenous metabolites in the daily diet to maintain the regeneration of the siphon in S. grandis. It is well established that several amino acids are converted into intermediates in the TCA cycle. Following activation of the anaplerotic process by autotomy, the concentrations of amino acids involved in this process change. In this study, glycine, cysteine, asparagine, and alanine were significantly decreased, whereas threonine levels were significantly increased after autotomy. Threonine is reversibly catalyzed to asparagine and allothreonine after autotomy of the siphon in S. grandis. Glutamine is also catalyzed to succinate via the glutathione metabolic pathway. Therefore, these pathways appear to be catalyzed to decrease the levels of the TCA cycle by autotomy.
Autotomy also affected carbohydrate metabolism in S. grandis. For energy production, glycolysis is initiated from glucose 6-phosphate, which is phosphorylated glucose. In the present study, glucose 6-phosphate levels significantly decreased after autotomy. Similarly, the levels of ADP-glucose, ADP-ribose, and 5′-phosphoribosyl ; the x-axis and y-axis represent enriched metabolic pathways and enrichment factors, respectively; rich factor refers to the ratio of differentially expressed metabolites to all metabolites annotated to this metabolic pathway; the larger the rich factor, the greater is the degree of enrichment. P-value, ranging from 0 to 0.05; the closer to 0, the more significant is the enrichment. www.nature.com/scientificreports/ amine were also significantly decreased after autotomy. Figure 8 shows that the concentrations of these metabolites were relatively lower than those of other metabolites involved in the glycolytic and purine and nucleotide sugar metabolic pathways. Additionally, there is a shortage of intermediate compounds and energy caused by the downregulation of succinate. The relatively low carbohydrate content observed in this study can be attributed to the glucose and glucose conjugates that break down maltose being converted into TCA cycle intermediates 41 . Therefore, such changes in sugar levels are expected to affect the generation of energy and intermediate compounds in order to maintain normal biological processes 21 . Glycose may also be a key factor in promoting the regeneration of the siphon of razor clams. In addition, autotomy affected the vitamin and cofactor metabolism of S. grandis, including dethiobiotin (vitamin B7), vitamin B6 and thiamine (vitamin B1). The dethiobiotin content decreased significantly after autotomy of the siphon. This is because cofactors play critical roles in the intermediate metabolism of gluconeogenesis, amino acid catabolism and fatty acid biosynthesis 42,43 . Hence, targeted metabolomics for the vitamin B-complex family should be considered to explore the effect of autotomy on the vitamins of S. grandis.